Lactoferrin is a multifunctional, non-heme iron-binding protein found in high concentration in milk, some mucosal secretions, and in specific granules of polymorphonuclear leukocytes. We know a great deal about lactoferrin structure, iron binding, effects on pathogenic agents and binding to various types of cells. What we really do not understand is the physiological role of the molecule, particularly in the milk and mammary gland. Lactoferrin provides 25% of the protein in human milk, yet formula-fed infants, who receive no lactoferrin in their milk, seemingly do well. The technical capacity to add lactoferrin to infant formula is rapidly being developed. The question is, is it necessary or beneficial and under what conditions? The present approach uses mice as a model system to investigate the physiological role of this elegant molecule. This model system has the advantage over dietary or formula supplementation that altered doses of lactoferrin are delivered to the neonate from the time of birth and in the natural, species specific milk. Our specific experimental approach will use transgenic technology in mice to provide critical tests of the dual hypotheses that: (1) The major roles of lactoferrin in neonatal development in the human are i) to regulate iron balance specifically by removing excess iron from the gastrointestinal tract, ii) to stimulate intestinal maturation and iii) to decrease growth of potential bacterial pathogens. (2) In the mammary gland itself lactoferrin protects against infection and inflammation during periods of secretory stasis, e.g. late pregnancy, involution and mastitis. To test these hypotheses we will first examine the effects of overexpression of human lactoferrin in mouse milk on neonatal development and iron balance using transgenic mice already available. We will then create transgenic mice overexpressing homologous lactoferrin in order to understand the species-specific effects of the molecule. Finally, we will develop technology to obtain mammary-specific inhibition of lactoferrin synthesis during lactogenesis and involution. In the absence of large supplies of active, recombinant lactoferrin the strategy outlined is the only one that can supply both human and homologous lactoferrin in large quantities to neonatal animals. If the inhibition approach is successful, we will in addition be able to define the effects of lactoferrin deficiency both in the mammary gland and in the neonate. The power of the transgenic techniques proposed are that they are mammary specific and address the long term physiology of lactoferrin n both mother and infant.